Allergy to peanuts and tree nuts is the most common food-related cause of severe anaphylaxis and death and, unlike other food allergies, typically persists into adulthood. Studies to date demonstrate at least two peanut characteristics that may contribute to its allergenicity: resistance to digestion and the ability of the major peanut allergen, Ara h1 to promote a Th2 response by binding to ICAM-1-grabbing non-integrin on dendritic cells. Our studies now demonstrate additional effects that are likely to contribute to strong allergenicity: water-soluble peanut extract (PE) causes shock and inflammatory cytokine production when administered to mice. Specifically, we demonstrate that: 1) complement, more specifically, C3a, and, to a lesser extent, stimulatory immunoglobulin (Ig) receptors contribute to PE-induced shock;2) PE-induced shock depends more on macrophages than on mast cells but is accompanied by mast cell activation;3) platelet activating factor (PAF) and, to a lesser extent, histamine contribute to PE-induced shock;4) TLR2, TLR4, MyD88, and the adaptive immune system are not required for PE-induced shock;5) PE acts synergistically with IgE-mediated mast cell degranulation to induce shock;and 6) PE acts through a C3-independent mechanism to induce TNF production and low level Th2 cytokine production but does not stimulate IFN-? production. These observations suggest three intriguing hypotheses that, together, offer a potential explanation for why peanuts are so allergenic: 1) peanuts have components that induce shock primarily by causing the production of complement- derived anaphylatoxins that induce macrophages and mast cells to produce PAF and histamine;2) the C3- related anaphylactoid response may act synergistically with peanut-induced IgE-mediated mast cell degranulation to induce the severe anaphylaxis experienced by some peanut-allergic patients;and 3) the inflammatory response stimulated by peanut components acts as an adjuvant that promotes the induction of a Th2 response to the major peanut allergens. We propose three aims to test these hypotheses and identify the molecule or molecules in peanuts that promote allergenicity. Studies in Aim 1 will use monoclonal antibodies generated against PE are to purify individual peanut macromolecules by affinity chromatography, mass spectroscopy to identify these molecules and in vivo studies to test their ability to induce shock and inflammatory cytokine production. Studies in Aim 2 will determine whether PE causes the generation of C3a in mouse and human plasma in vitro and will use complement factor B- and C4-deficient mice to identify the pathway by which PE activates C3. Studies in Aim 3 will: 1) immunize mice to determine whether proteins purified from PE that induce TNF production and/or complement activation have adjuvant activity;2) determine whether TNF production is stimulated by peanut proteins that are toll-like receptor ligands, oxidants, and/or lectins;and 3) use flow cytometric approaches and techniques for depleting specific cell types in vivo to identify the cells that are induced by PE to produce TNF. RFA-AI-07-032/Finkelman, Fred D. Identification of Proteins Responsible For Peanut Allergenicity This application is responsive to the Exploratory Investigations in Food Allergy RFA by using ex vivo studies and in vivo mouse model studies to investigate the pathogenesis of food allergy. Specifically, we are studying why peanuts, which now cause allergy in 0.8% of Americans, are so allergenic. Elucidation of the special characteristics of peanuts and tree nuts that contribute to their allergenicity may provide clues to food allergy pathogenesis in general, suggest ways to prevent and treat this disorder, and suggest ways to genetically modify peanuts and tree nuts to make them less allergenic.